Oligonucleotide analogues

ABSTRACT

An oligonucleotide analogue having 10 to 200 natural and/or synthetic nucleoside units, linked by internucleoside linkages, at least one of the internucleoside linkages being of formula                    
     where the indicated methylene group is attached to a 3′ carbon atom of a nucleoside, the indicated oxygen atom is attached to a 5′-carbon atom of an adjacent nucleside, R 1  is hydrogen, hydroxy, O—, thiol, S—, —NH 2  or a group of formula R 1a , —OR 1a , —SR 1a , —NHR 1b , or —NR 1b R 1c  wherein R 1a  is an unsubstituted or substituted C 1  to C 10  alkyl, C 2  to C 10  alkenyl, C 3  to C 8  cycloalkyl, C 6  to C 10  aryl or C 7  to C 13  aralkyl group; and R 1b  and R 1c  are each independently an unsubstituted or substituted C 1  to C 10  alkyl, C 2  to C 10  alkenyl, C 3  to C 8  cycloalkyl, C 6  to C 10  aryl or C 7  to C 13  aralkyl group or R 1b  and R 1c  together with the nitrogen atom to which they are attached denote a five- or six-membered heterocyclic ring, and X is oxygen or sulfur.

This invention relates to oligonucleotide analogues, their preparationand their use.

In accordance with the invention, oligonucleotide analogues can beprepared which have good hybridisation properties to single- anddouble-stranded nucleic acids, RNase H-activating properties, goodhydrolytic stability and good stability towards cleavage by nucleases,facilitating their use as inhibitors of gene expression, for example byantisense interaction, and as pharmaceuticals in the treatment ofdiseases such as cancer and viruses such as influenza, herpes and HIV.

Accordingly, the present invention provides an oligonucleotide analoguehaving 10 to 200 natural and/or synthetic nucleoside units linked byinternucleoside linkages, at least one of the internucleoside linkagesbeing of formula

where the indicated methylene group is attached to a 3′ carbon atom of anucleoside, the indicated oxygen atom is attached to a 5′ carbon atom ofan adjacent nucleoside, R¹ is hydrogen, hydroxy, O⁻, thiol, S⁻, —NH₂ ora group of formula R¹ _(a), —OR¹ _(a), —SR¹ _(a), —NHR¹ _(b) or —NR¹_(b)R¹ _(c) where R¹ _(a) is an unsubstituted or substituted C₁ to C₁₀alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ toC₁₃ aralkyl group and R¹ _(b) and R¹ _(c) are each independently anunsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ toC₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group or R¹ _(b) andR¹ _(c) together with the nitrogen atom to which they are attacheddenote a five- or six-membered hetercyclic ring, and X is oxygen orsulphur.

The number of nucleoside units in the oligonucleotide analogue may vary,for example from 15 to 100, according to the nature of the nucleic acidsequence to which the oligonucleotide analogue is targeted. Preferably,the oligonucleotide analogue has 15 to 40, especially 15 to 25nucleoside units. The oligonucleotide analogue may more preferably have15 to 20 nucleoside units for certain targets, 20 to 25 nucleoside unitsfor other targets, 18 to 25 nucleoside units for further targets and 18to 22 nucleoside units for yet further targets.

In an oligonucleotide analogue of the invention, the number ofinternucleoside linkages of formula I may vary according to theproperties desired. For example, for some purposes one internucleosidelinkage of formula I may suffice, while for other purposes all theinternucleoside linkages may be of formula I and may be the same ordifferent. For most purposes, up to 75%, for example up to 50%,particularly up to 25%, of the internucleoside linkages may be offormula I.

In some embodiments of the invention, at least two consecutiveinternucleoside linkages, for example two, three, four, five or sixconsecutive internucleoside linkages, which may be the same ordifferent, in the oligonucleotide analogue are of formula I. There maybe such a sequence of consecutive internucleoside linkages at each endof the oligonucleotide analogue; more usually, there is one suchsequence of consecutive internucleoside linkages of formula I betweensequences of nucleosides having other internucleoside linkages. In otherembodiments of the invention having two or more internucleoside linkagesof formula I, internucleoside linkages of formula I may alternate withother internucleoside linkages, for example along the whole length ofthe oligonucleotide analogue or in a region at one or both ends of theoligonucleotide analogue, or in a region in the middle of theoligonucleotide analogue.

In embodiments of the invention where not all of the internucleosidelinkages are of formula I, the remaining internucleoside linkages may benatural phosphodiester linkages or other synthetic substitutes thereforsuch as phosphorothioate, phosphorodithioate, alkylphosphonate(—O—P(O)(R)O—), phosphoramidate, short chain alkyl, cycloalkyl, shortchain heteroatomic, —NHCOCH₂—, —CH₂NHCO—, —CONHCH₂—, —CH₂CONH—,—CH₂NHO—, —CH₂N(CH₃)O—, —CH₂ON(CH₃)—, —CH₂N(CH₃)N(CH₃)— or —ON(CH₃) CH₂—linkages, or combinations of two or more such linkages. Preferably, theremaining internucleoside linkages are phosphodiester, phosphorothioateor phosphorodithioate linkages or a mixture of two or more of thesethree types, particularly phosphodiester, phosphorothioate or a mixtureof phosphodiester and phosphorothioate linkages. In certain especiallypreferred embodiments the remaining internucleoside linkages arephosphorothioate linkages.

Preferably, not more than 50% of the internucleoside linkages arephosphorothioate linkages.

In certain embodiments of the invention, the oligonucleotide comprises aregion having phosphodiester and/or phosphorothioate and/orphosphorodithioate internucleoside linkages between two regions havinginternucleoside linkages of formula I, or a mixture thereof withphosphorothioate or phosphodiester linkages, particularly a regionhaving phosphorothioate linkages between two regions havinginternucleoside linkages of formula I or a mixture thereof withphosphorothioate or phosphodiester linkages.

In some especially preferred embodiments, the oligonucleotide analogueof the invention comprises a region of at least 6 nucleosides linked byphosphorothioate linkages between two regions having nucleosides linkedonly by internucleoside linkages of formula I.

In oligonucleotide analogues of the invention, the nucleoside units maybe natural or synthetic nucleosides having a purine or pyrimidine basesuch as adenine, guanine, cytosine, thymine or uracil, or an analogue ofthese bases such as 2-aminoadenine, 6-hydroxypurine, 5-methylcytosine,5-propynylcytosine, 5-fluorouracil, 5-propynyluracil or dihydrouracil,attached to the I′ carbon atom of a furanose sugar. As is wellunderstood by those skilled in the art, when the oligonucleotides arefor use in antisense applications, the sequence of nucleosides is chosento be complementary to a target RNA sequence. For example, theoligonucleotide analogue of the invention may be complementary to aregion of mRNA for human c-raf kinase, in which case, a preferredsequence is

5′-TCC CGC CTG TGA CAT GCA TT-3′ SEQ ID NO:1.

described as Seq. ID No. 8 in WO 95/32987 or the oligonucleotideanalogue of the invention may be complementary to a region of mRNA forhuman PKC-α, in which case a preferred sequence is

5′-GTT CTC GCT GGT GAG TTT CA-3′ SEQ ID NO:2

described as Seq. ID No. 2 in WO 95/02069.

In some oligonucleotide analogues of the invention, at least onenucleoside is modified at the 2′ position thereof, for example toincrease binding affinity for a given target and/or to increase nucleaseresistance. All of the nucleosides may be so modified, or up to 80%, forexample up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to20%, or up to 10%, of the nucleosides, may be so modified. Examples of2′ modifying atoms and groups, i.e. atoms or groups which may beattached to the 2′ position of a nucleoside in place of a hydrogen atomor hydroxy group to effect a modification, include halogen atoms such asfluorine, chlorine and bromine atoms; C₁ to C₁₀ unsubstituted orsubstituted alkyl groups such as methyl, trifluoromethyl, ethyl, propyl,butyl, pentyl, hexyl, octyl or decyl; C₆ to C₁₀ aryl groups such asphenyl, tolyl or xylyl; C₇ to C₁₃ aralkyl groups such as benzyl; amino,C₁ to C₁₀ alkyl amino such as methylamino, ethylamino or octylamino; C₁to C₁₀ alkylthio such as methylthio, ethylthio or octylthio; azide;nitrate; nitrite; cyanide; cyanate; methanesulphonate; C₁ to C₁₀aminoalkylamino; a group of formula —OR² where R² is a C₁ to C₁₀aliphatic group; substituted silyl; an RNA cleaving group; a cholesterylgroup; a conjugate; a reporter group; an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide; and agroup for improving the pharmacodynamic properties of anoligonucleotide.

Preferred modifying atoms and groups at the 2′ position are halogenatoms, especially fluorine, and a group of formula —OR² where R² is a C₁to C₁₀ aliphatic group, which may be an unsubstituted or substituted C₁to C₁₀ alkyl group such as methyl, ethyl, isopropyl, butyl, hexyl,octyl, decyl, trifluoromethyl, ethoxyethyl, methoxyethyl, orbutoxyethyl, or a C₂ to C₆ alkenyl group such as vinyl, allyl ormethallyl. Particularly preferred modifying atoms and groups arefluorine and groups of formula —OR² where R² is an unsubstituted orsubstituted C₁ to C₁₀ alkyl group, preferably C₁ to C₄ alkyl, C₁ to C₄alkoxy-substituted C₁ to C₄ alkyl or a group of formula—(CH₂CH₂O)—_(n)R³ when R³ is methyl or ethyl and n is 2 to 4. Especiallypreferred groups of formula —OR² are those where R² is methyl, ethyl,methoxyethyl, ethoxyethyl or a group of formula —(CH₂CH₂O)—₃CH₃.

When nucleosides modified at the 2′ position are present, anoligonucleotide analogue of the invention may have, for example, atleast two consecutive nucleosides modified at the 2′ position and linkedby phosphodiester internucleoside linkages and/or it may have aninternucleoside linkage of formula I between a nucleoside unmodified atthe 2′ position and a 5′ carbon atom of a nucleoside modified at the 2′position.

As is also well understood by those skilled in the art, the terminalnucleosides in the oligonucleotide analogue may have free 5′ and 3′hydroxy groups respectively or may have either or both of these hydroxygroups replaced by a modifying group, for example a phosphate, thiol,alkylthio, thioalkyl, thiophosphate, aminoalkyl, acridinyl, cholesterylor fluoresceinyl group.

In linkages of formula I, R¹ _(a), R¹ _(b) or R¹ _(c) as a substitutedalkyl, alkenyl, cycloalkyl, aryl or aralkyl group may be substituted,for example, by hydroxy, C₁ to C₄ alkoxy, halogen (preferably chlorineor fluorine), cyano, tri(C₁-C₁₅ hydrocarbyl)silyl, or primary, secondaryor tertiary amino.

In a linkage of formula I, where R¹ is R¹ _(a), —OR¹ _(a) or —SR¹ _(a),R¹ _(a) as C₁ to C₁₀ alkyl may be, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, pentyl, hexyl, octyl, nonyl or decyl,preferably C₁ to C₄ alkyl; R¹ _(a) as C₂ to C₁₀ alkenyl may be vinyl,allyl, methallyl, 1-propenyl, isopropenyl, 2-butenyl, 1-butenyl,isobutenyl, pentenyl, hexenyl, octenyl or decenyl, preferably C₂ to C₅alkenyl; R¹ _(a) as C₃ to C₈ cycloalkyl may be, for example,cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl,methylcyclohexyl, dimethylcyclohexyl, cycloheptyl or cyclooctyl,preferably C₅ to C₈ cycloalkyl; R¹ _(a) as C₆ to C₁₀ aryl may be, forexample, phenyl, o-tolyl, m-tolyl, p-tolyl, o-xylyl, m-xylyl, p-xylyl ornaphthyl, preferably C₆ to C₈ aryl; R¹ _(a) as C₇ to C₁₃ aralkyl may be,for example, benzyl, 4-methylbenzyl, 2-phenylethyl, 2-phenylpropyl,3-phenylpropyl or diphenylmethyl, preferably C₇ to C₉ aralkyl. R¹ as—NHR¹ _(b) may be C₁ to C₁₀ alkylamino, for example, methylamino,ethylamino, isopropylamino, butylamino, pentylamino, hexylamino,octylamino or decylamino, preferably C₁ to C₄ alkylamino; C₂ to C₁₀alkenylamino, for example allylamino, methallylamino, 1-propenylamino,isopropenylamino, isobutenylamino, hexenylamino, octenylamino ordecenylamino, preferably C₃ to C₅ alkenylamino; C₃ to C₈cycloalkylamino, for example, cyclopropylamino, cyclobutylamino,cyclopentylamino, cyclohexylamino, cycloheptylamino, cyclooctylamino ordimethylcyclohexylamino, preferably C₅ to C₈ cycloalkylamino; C₆ to C₁₀arylamino, for example phenylamino, ortho-, meta- or para-tolylamino,ortho-, meta- or para-xylylamino or naphthylamino, preferably C₆ to C₈arylamino; C₇ to C₁₃ aralkylamino, for example benzylamino,4-methylbenzylamino, 2-phenylethylamino, 3-phenylpropylamino ordiphenylmethylamino, preferably C₇ to C₉ aralkylamino. R¹ as —NR¹ _(b)R¹_(c) may be di(C₁ to C₁₀ alkyl)amino, for example, dimethylamino,diethylamino, methylethylamino, diisopropylamino, dibutylamino ordioctylamino, preferably di(C₁ to C₄ alkyl) amino; N,N-di(C₂-C₁₀alkenyl)amino, for example diallylamino, dimethallylamino,allymethallylamino, dipropenylamino, dibutenylamino, dipentenylamino,dihexenylamino, dioctenylamino or didecenylamino, preferably di(C₃-C₅alkenyl)amino; N,N-di(C₃-C₈ cycloalkyl)amino, for exampledicyclopropylamino, cyclopropylcyclopentylamino, dicyclobutylamino,dicyclopentyl amino, dicyclohexylamino, dicycloheptylamino ordicyclooctylamino, preferably N,N-di(C₅-C₈ cycloalkyl)amino; N-C₃-C₈cycloalkyl-N-C₁-C₁₀ alkylamino, for example N-cyclopentyl-N-methylamino,N-cyclopentyl-N-ethylamino, N-cyclohexyl-N-methylamino,N-cyclohexyl-N-ethylamino, preferably N-(C₅-C₈ cycloalkyl)-N-C₁-C₄alkylamino; N-C₆-C₁₀-aryl-N-C₁-C₁₀ alkylamino, preferablyN-C₆-C₈-aryl-C₁-C₄ alkylamino, for example N-phenyl-N-methylamino,N-tolyl-N-methylamino or N-phenyl-N-ethylamino; N, N-di(C₇-C₁₃aralkyl)amino, for example dibenzylamine, di(4-methylbenzyl)amine,di(phenylethyl)amino or di(phenylpropyl)amino, preferably N,N-di(C₇-C₉aralkyl)amino; or N-C₇-C₁₃ aralkyl-N-C₁-C₁₀ alkylamino, preferablyN-C₇-C₉ aralkyl-N-C₁-C₄ alkylamino, for example N-benzyl-N-methylaminoor N-benzyl-N-ethylamino or a radical of a five- or six-memberedN-heterocycle linked through the nitrogen atom to the indicatedphosphorus atom in formula I, for example 1-pyrrolidinyl, 1-piperidyl,1-piperazinyl or morpholino. Any of the above groups may beunsubstituted or substituted as hereinbefore described.

In certain preferred embodiments, R¹ is hydrogen, hydroxy, O³¹ , SH, S³¹, an unsubstituted or substituted C₁ to C₄ alkyl or phenyl group, agroup of formula —OR¹ _(a) where R¹ _(a) is an unsubstituted orsubstituted C₁ to C₄ alkyl, C₃ to C₅ alkenyl, C₅ to C₈ cycloalkyl or C₇to C₉ aralkyl group, or a group of formula —SR¹ _(a) where R¹ _(a) is anunsubstituted or substituted C₁ to C₄ alkyl or phenyl group, optionalsubstituents being as hereinbefore described. In some especiallypreferred embodiments, R¹ is hydrogen, hydroxy, O³¹ , SH, S³¹ , methoxy,ethoxy or 2-cyanoethoxy.

Where R¹ in formula I is O⁻, the oligonucleotide analogue of theinvention may be in the form of a pharmaceutically acceptable salt, forexample metal salt, preferably an alkali metal salt, or an unsubstitutedor substituted ammonium salt, for example a mono-, di- or tri-C₁ to C₁₀alkyl- or hydroxyalkyl-ammonium salt, a N-ethylpiperidinylium salt or aN,N¹-dimethylpiperazinylium salt. In especially preferred embodimentswhere R¹ is O⁻, the oligonucleotide analogue is in the form of thesodium or ammonium salt.

Where the phosphorus atom in formula I is a chiral centre, differencesmay be observed in hybridisation and nuclease resistance properties andin biological efficacy depending on the stereochemistry at phosphorus.

An oligonucleotide analogue of the invention may be represented by theformula V—LV—L_(n)V where n is a number from 8 to 198, each V isindependently a residue of a natural or synthetic nucleoside, each ofthe n+2 residues V being the same as, or different from, an adjacentresidue V, and each L is an internucleoside linkage, each of the n+1linkages L being the same as, or different from, an adjacent linkage L,at least one L being of formula I.

The present invention also provides a method of preparing anoligonucleotide analogue having at least one internucleoside linkage offormula I, for example an oligonucleotide having 2 to 200 nucleosideunits, such as an oligonucleotide analogue as hereinbefore described,which comprises (i) carrying out a coupling reaction or successivecoupling reactions between (A) a natural or synthetic nucleoside oroligonucleotide having a 5′-hydroxyl group and (B) a natural orsynthetic nucleoside or dinucleotide having at the 3′-position thereof agroup reactive with said 5′-hydroxyl group until an oligonucleotidehaving the desired number of nucleosides is obtained, in at least one ofsaid coupling reactions (B) being a nucleoside of formula

where B¹ is a nucleoside base radical, R⁴ is a hydroxy-protecting group,R⁵ is hydrogen, hydroxy or a 2′ modifying atom or group, M⁺ is a metalor unsubstituted or substituted ammonium ion or a cation of aheterocyclic base such as pyrrolidine, piperidine, N-ethylpiperidine,N,N¹-dimethylpiperazine, morpholine or 1,8diazabicyclo[5.4.0]undec-7-ene (DBU) and X is oxygen or sulphur, andbeing reacted with (A) in the presence of a sterically hindered organicacid halide or anhydride to form an oligonucleotide analogue having aphosphinate internucleoside linkage of formula

where X is oxygen or sulphur, and (ii)(a) oxidising the phosphinatelinkage or (b) sulphurising the phosphinate linkage, or (c) reacting thephosphinate linkage with a compound of formula R¹ _(a)Y where R¹ _(a) isas hereinbefore defined and Y is a leaving atom or group or (d)oxidising and reacting the phosphinate linkage with an alcohol offormula R¹ _(a)OH or an amine of formula R¹ _(b)NH₂ or R¹ _(b)R¹ _(c) NHwhere R¹ _(a), R¹ _(b) and R¹ _(c) are as hereinbefore defined, or (e)silylating the phosphinate linkage and reacting the silylated linkagewith a thioalkylating or thioarylating agent to give a phosphinatelinkage of formula I where R¹ is —SR¹ _(a) where R¹ _(a) is ashereinbefore defined.

The hereinbefore defined method may be carried out in solution or on asolid carrier, for example using known procedures for oligonucleotidesynthesis. The oligonucleotide analogue obtained by this method may befurther reacted to replace the protecting group R⁴ by hydrogen or, whereR⁴ is on a terminal nucleoside in the oligonucleotide analogue, by a 5′modifying group as hereinbefore described.

Oligonucleotide analogues of the invention may be prepared by solidphase synthesis, for example using H-phosphonate, phosphotriester orphosphoramidite methods, or a mixture of two or more thereof, forexample automatically using commercially available nucleic acidsynthesisers. A solid phase synthetic method may comprise carrying outsuccessive coupling reactions (i) as hereinbefore described and step(ii) as hereinbefore described with the nucleoside or oligonucleotide(A) attached to the solid support, then (iii) detaching theoligonucleotide from the solid support and removing protecting groups togive an oligonucleotide having a terminal 5′ free hydroxyl group and(iv) optionally reacting the 5′ free hydroxyl group to introduce amodifying group at the terminal 5′ position. In the nucleoside offormula II, B¹ may be a purine or pyrimidine base or analogue thereof ashereinbefore described. Compounds where B¹ is a natural nucleoside base,more preferably pyrimidine base, especially thymine, are preferred. R⁴may be any hydroxy-protecting group capable of protecting the 5′hydroxyl group against undesired reaction. Such groups are well knownand include C₁ to C₁₀ aliphatic, e.g. alkyl, groups; C₃ to C₈cycloaliphatic, e.g. cycloalkyl, groups; C₆ to C₁₀ aromatic, e.g. aryl,groups; C₇ to C₄₀ araliphatic, e.g. aralkyl or C₁ to C₄alkoxy-substituted aralkyl, groups; groups of formula —COR⁶ or —SO₂R⁶where R⁶ is a C₁ to C₁₀ aliphatic group, a C₃ to C₈ cycloaliphaticgroup, a C₆ to C₁₀ aromatic group or a C₇ to C₄₀ araliphatic group; andtri(C₁-C₁₅ hydrocarbyl)silyl groups. Preferably R⁴ is a 5′ protectinggroup conventionally used in oligonucleotide synthesis, especially amethoxytrityl, dimethoxytrityl or tris tert-butyltrityl group. R⁵ as a2′ modifying atom or group may be such an atom or group as hereinbeforedescribed; preferably R⁵ is hydrogen. M⁺ may be, for example, an alkalimetal ion or, preferably, an unsubstituted ammonium ion, a mono-di- ortri-C₁ to C₁₀ alkyl- or hydroxyalkyl-ammonium ion or a cation of aheterocyclic base such as pyrrolidine, piperidine, N-ethylpiperidine,N,N-dimethylpiperazine, morpholine or DBU. An especially preferred M⁺ isa triethylammonium ion.

Preferred stereoisomers of nucleosides of formula II are those offormula

where B¹, R⁴, R⁵, X and M⁺ are as hereinbefore defined.

Nucleosides of formula II may be prepared by a) reacting a compound offormula

where B¹ and R⁴ are as hereinbefore defined, R⁵ _(a) is hydrogen,fluorine or —OR² where R² is as hereinbefore defined and L is a leavingatom or group, preferably an iodine atom, with ethyl (1,1-diethoxyethyl)phosphinate in the presence of a base such as potassiumbis(trimethylsilyl)amide in tetrahydrofuran (THF) at −80° C. to 40° C.to give a compound of formula

where B¹ and R⁵ _(a) are as hereinbefore defined, b) reacting thecompound of formula IV with trimethylsilyl chloride in chloroformcontaining 1% ethanol under argon at ambient temperature to replace theprotecting ketal group attached to phosphorus by hydrogen, c) reactingthe product from b) with acetone in the presence of titanium (IV)isopropoxide in dry THF at ambient temperature to give a compound offormula

d) reacting the compound of formula V with tetra-n-butylammoniumfluoride and acetic acid in THF at ambient temperature to remove thetert-butyldiphenylsilyl protecting group, e) reacting the resulting5′-hydroxy-containing compound with a compound of formula R⁴Y, where R⁴is as hereinbefore defined and Y is halogen, in the presence of anorganic base to give a compound of formula

where B¹, R⁴ and R⁵ _(a) are as hereinbefore defined, f) reacting thecompound of formula VI with an alkali metal methoxide in anhydrousmethanol, or with DBU in water, at ambient temperature to remove theethyl group and replace the —C(CH₃)₂ OH group by hydrogen, g) ifdesired, treating the product with ammonia or an amine to form thecorresponding unsubstituted or substituted ammonium salt, and h) ifdesired, sulphurising the product to give a nucleoside of formula II inwhich X is sulphur, for example by reaction with pivaloylchloridefollowed by (CH₃)₃ Si—S—Si(CH₃)₃ using the procedure described in J.Org. Chem. 1995, 60, 8241.

The compounds of formulae IV, V or VI may be reacted to introduce, orintroduce a different, modifying atom or group R⁵ at the 2′ positionusing, for example, a conventional procedure for introducing such a 2′modifying atom or group into a nucleoside.

Compounds of formula III may be prepared by reducing an aldehyde offormula

where B¹ and R⁵ _(a) are as hereinbefore defined, prepared according tothe method described in WO 92/20823, to the corresponding alcohol byreaction with NaBH₄ in anhydrous ethanol at ambient temperature andreacting the alcohol with methyltriphenoxyphosphonium iodide in thepresence of 2,6-lutidine in dry dimethyl formamide at 0° C. to 30° C.

Ethyl(1,1-diethoxyethyl)phosphinate may be prepared as described in EP 0307 362.

In a typical procedure using solid phase synthesis, a natural orsynthetic nucleoside having a protected 5′ hydroxyl group is covalentlylinked at the 3′ position to an inert silica-based support, such ascontrolled pore glass (CPG), containing long chain alkylamino groups,using a linker such as succinic anhydride to give a 3′-terminalnucleoside attached to the solid support. The solid support may alsocontain groups to act as 3′ terminal modifying groups for the desiredoligonucleotide. The protecting group, for example a dimethoxytritylgroup, on the 5′ hydroxy of the attached terminal nucleoside is thenremoved to give a free 5′ hydroxy group. The terminal nucleoside is thencoupled with a natural or synthetic nucleoside or dinucleotide (B)having a protected, e.g. dimethoxytrityl-protected, 5′ hydroxyl groupand, at the 3′ position a group which is reactive with, or activatableto be reactive with, the 5′ free hydroxyl group on the terminalnucleoside to give a dimeric oligonucleotide (or, where (B) is adinucleotide, a trimeric oligonucleotide) attached to the solid support.After the 5′ protecting group on the attached oligonucleotide has beenremoved, the reaction cycle with a natural or synthetic 5′ protectednucleoside or dinucleotide (B) having a 3′ reactive group is repeateduntil an oligonucleotide having the desired number of nucleosides hasbeen synthesised.

For the formation of internucleoside linkages other than those offormula I, the reactive group, or the group activatable to be reactive,at the 3′ position of the nucleoside or dinucleotide (B) is chosen inaccordance with conventional oligonucleotide synthesis procedures andmay be, for example, an H-phosphonate group, a phosphoramidite group ora phosphodiester group. The coupling reactions and, where necessary,subsequent oxidation, sulphurisation or other treatment, to form theseinternucleoside linkages, for example phosphotriester, phosphorothioateor phosphorodithioate linkages, may be carried out using conventionalprocedures.

In preparing an oligonucleotide analogue of the invention, at least oneof the coupling reactions is carried out using as (B) a nucleoside offormula II, which is reacted with the nucleoside or oligonucleotide (A),which in solid phase synthesis is attached to the solid support, in thepresence of a sterically hindered organic acid halide or anhydride, forexample pivaloyl chloride, adamantoyl chloride,2,4,6-triisopropylbenzenesulphonyl chloride, diphenylphosphinicchloride, bis(2-oxo-3-oxazolidinyl) phosphinic chloride,2-chloro-2-oxo-5,5-dimethyl-1,3,2-dioxaphosphinane orbis(pentafluorophenyl) anhydride. Preferably, the reaction is carriedout in the presence of a heterocyclic base having a tertiary nitrogenatom in the ring or an oxide of such a base, for example, pyridine,quinoline, N-methylimidazole or pyridine-N-oxide and, especially, anorganic solvent such as acetonitrile. The coupling reaction may becarried out at ambient or moderately elevated temperatures, for exampleup to 50° C.

The phosphinate internucleoside linkage of formula IA formed by acoupling reaction using as (B) a nucleoside of formula II may beoxidised or sulphurised before the next coupling reaction is carried outor, preferably, after an oligonucleotide analogue with the desirednumber of nucleosides has been synthesised, when it may be oxidised orsulphurised together with one or more other phosphinate internucleosidelinkages formed by other coupling reactions using a nucleoside offormula II or phosphite linkages formed by coupling reactions using a 3′H phosphonate-substituted nucleoside. Oxidation (ii)(a) may be effectedby treatment with iodine and water, or with tert-butyl hydroperoxide,for example using conventional procedures for oxidation of phosphiteinternucleoside linkages. Sulphurisation (ii)(b) may be effected bytreatment with sulphur in the presence of a tertiary amine in an organicsolvent, usually carbon disulphide, for example using known procedures.

The reaction (ii)(c) of the phosphinate internucleoside linkage offormula IA with a compound of formula R¹ _(a)Y where R¹ _(a) and Y areas hereinbefore defined, Y preferably being a halogen atom or atrifluoromethanesulphonate group, may be carried out, where R¹ _(a) isalkyl, cycloalkyl or aralkyl, using known alkylation procedures, forexample by reacting the phosphinate linkage of formula IA with R¹ _(a)Ywhere Y is halogen in the presence of a strong base such as sodiumhydride. Where R¹ _(a)Y is an alkenyl or aryl halide or triflate, thereaction between the phosphinate linkage and R¹ _(a)Y may be carried outusing known procedures, for example in the presence of a palladiumcatalyst such as Pd(PPh₃)₄ and a tertiary amine, for example asdescribed by Y.Xu et al, Tetrahedron Lett., 30, 949(1989) or K. S.Petrakis et al, J. Am. Chem. Soc., 109, 2831 (1987).

The oxidative reaction (ii)(d) of the phosphinate internucleosidelinkage of formula IA with an alcohol of formula R¹ _(a)OH, before thenext coupling reaction or after an oligonucleotide with the desirednumber of nucleosides has been synthesised, may be carried out byreaction with an oxidant such as iodine, carbon tetrachloride orbromotrichloromethane in the presence of the alcohol R¹ _(a)OH and abase such as pyridine, for example under conventional conditions foroxidation of phosphite internucleoside linkages.

The oxidative reaction (ii)(d) of the phosphinate internucleosidelinkage of formula IA may be effected, before the next coupling reactionor after an oligonucleotide with the desired number of nucleosides hasbeen synthesised, with an amine of formula R¹ _(b)NH₂ or R¹ _(b)R¹_(c)NH where R¹ _(b) and R¹ _(c) are as hereinbefore defined, and carbontetrachloride or bromotrichloromethane or iodine to give anoligonucleotide analogue of the invention in which R¹ is —NHR¹ _(b) or—NR¹ _(b)R¹ _(c) respectively. The reaction may be carried out usingknown conditions and procedures for Atherton-Todd reactions.

The reaction (ii)(e) of the phosphinate linkage of formula IA may becarried out by silylating the linkage using known silylation procedures,for example using a trialkylsilyl halide and a base such astriethylamine, and reacting the silylated linkage with a thioalkylatingor thioarylating agent such as a thiosulphonate of formula ArSO₂SR¹ _(a)where R¹ _(a) is as hereinbefore defined and Ar is an aromatic groupsuch as phenyl or tolyl. Suitable procedures for this reaction aredescribed by W. K. D. Brill, Tetrahedron Lett, 36, 703 (1995).

It will be apparent to those skilled in the art that in reacting thephosphinate linkage of formula IA to replace the hydrogen atom attachedto phosphorus by a group R¹ having a reactive substituent such as amino,the substituent should be protected during the reaction to introduce R¹if it is reactive under the conditions of that reaction and subsequentlydeprotected.

When an oligonucleotide analogue having the desired number ofnucleosides has been synthesised on a solid support, it is detached fromthe solid support, for example using conventional methods such astreatment with concentrated aqueous ammonia, which treatment alsoremoves a protecting group which may have been present on an exocyclicnitrogen atom in one or more of the nucleosides used in the synthesis ofthe oligonucleotide, before or after treatment to removehydroxy-protecting groups such as dimethoxytrityl groups, which may alsobe carried out using conventional methods, for example by treatment withan aqueous organic acid such as trifluoroacetic acid.

Before or after detachment of the oligonucleotide from the solidsupport, the terminal 5′ hydroxyl generated on deprotection can bereacted to introduce a 5′ terminal modifying group, such as a phosphategroup or other 5′ modifying group as hereinbefore described, for exampleusing the procedures described by Beaucage and lyer, Tetrahedron 49,1925-63 (1993).

In a modification of the synthetic method hereinbefore described, thenucleoside of formula II may be replaced by a dinucleotide of formula

where B¹, R¹, R⁴ and R⁵ are as hereinbefore defined, B² is a nucleosidebase radical, which may be a radical of a natural or syntheticnucleoside base as hereinbefore described for B¹, R⁶ is hydrogen,hydroxy or a 2′ modifying atom or group as hereinbefore defined for R⁵,and R⁷ is a group reactive with, or activatable to be reactive with, a5′ hydroxyl group in a nucleoside.

In this modification, the group R⁷O in the dinucleotide of formula VIIImay be a H-phosphonate group, in which case the dinucleotide of formulaVII may be reacted with the nucleoside or oligonucleotide (A), which insolid phase synthesis is attached to the solid support, in the presenceof a sterically hindered organic acid halide, for example usingconventional procedures for oligonucleotide synthesis using 3′H-phosphonates, to form a phosphite internucleoside linkage which maythen be oxidised, sulphurised or reacted with a compound of formula R¹_(a)Y or subjected to another of the reactions (ii)(a) to (ii)(e) ashereinbefore described for the phosphinate internucleoside linkage offormula IA formed by reaction of the nucleoside of formula II with (A).

In this modification, the group R⁷O in the dinucleotide of formula VIIImay alternatively be a phosphoramidite group, in which case thedinucleotide of formula VIII may be reacted with the nucleoside oroligonucleotide (A), for example using conventional procedures foroligonucleotide synthesis using 3′ phosphoramidites.

In another alternative embodiment of this modification, the group R⁷O inthe dinucleotide of formula VIII may be a phosphodiester group, in whichcase the dinucleotide of formula VIII may be reacted with the nucleosideor oligonucleotide (A), for example using conventional procedures foroligonucleotide synthesis using 3′ phosphodiesters.

Dinucleotides of formula VIII may be prepared by reacting a nucleosideof formula II with a nucleoside of formula

where B² and R⁶ are as hereinbefore defined and R⁸ is ahydroxy-protecting group, in the presence of a dehydrating couplingreagent e.g. a carbodiimide or a sterically hindered organic acid halideor anhydride, to give a dinucleotide of formula

and, optionally after subjecting the internucleoside linkage in formulaX to any of reactions (ii)(a) to (ii)(e) as hereinbefore described,converting the R⁸O— group into a R⁷O— group.

The hydroxy-protecting group R⁸ may be chosen from groups hereinbeforespecified for R⁴. Preferably R⁸ is a 3′ protecting group conventionallyused in nucleoside chemistry, especially a tert-butyldiphenylsilylgroup.

Nucleosides of formula IX are 3′ protected natural or syntheticnucleosides which may have hydrogen, hydroxy or a 2′ modifying atom orgroup at the 2′ position. Such nucleosides are known or may be preparedby known methods.

The reaction between the nucleoside of formula II and the nucleoside offormula IX in the presence of a sterically hindered organic acid halideis preferably carried out in the presence of a heterocyclic base oroxide thereof and an organic solvent as hereinbefore described for thereaction of the nucleoside of formula II with the nucleoside oroligonucleotide (A).

The conversion of the group R⁸O— into a group R⁷O— where R⁸ and R⁷ areas hereinbefore defined may be carried out using conventional methodsfor converting a protected 3′ hydroxyl group into a group reactive with,or activatable to be reactive with, a 5′ hydroxyl group, such as aH-phosphonate, phosphoramidite or phosphodiester group. For example, theprotecting group R⁸ may be removed to generate a free 3′ hydroxyl, whichmay then be reacted with an aliphatic bis(N,N-dialkyl)phosphoramiditesuch as 2-cyanoethyl bis(N,N-diisopropyl)phosphordiamidite to form a 3′phosphoramidite group.

Dinucleotides of formulae VIII where one or each of R⁵ and R⁶ is 2′modifying atom or group as hereinbefore defined, particularly a group offormula —OR² as hereinbefore defined, are novel. Dinucleotides offormula X are novel. Thus the invention also provides noveldinucleotides of formula

where B¹, B², R¹, R⁴ are as hereinbefore defined, R⁵ and R⁶ are ashereinbefore defined except that where R¹ is other than hydrogen atleast one of R⁵ and R⁶ is a 2′ modifying atom or group as hereinbeforedefined, and R⁹ is R⁷ or R⁸ as hereinbefore defined, especially thosewhere R⁵ is hydrogen or hydroxy and R⁶ is a 2′ modifying atom or groupas hereinbefore defined, particularly a group of formula —OR² ashereinbefore defined.

Dinucleotides of formula VIII where R¹ is C₁ to C₁₀ alkoxy may also beprepared by reacting a nucleoside of formula

where B¹, R⁴ and R⁵ are as hereinbefore defined, with a nucleoside offormula IX in the presence of a tertiary amine such asdimethylaminopyridine and a dehydrating agent such asdicyclohexylcarbodiimide (DCC), to give a dinucleotide of formula X,which is then treated as hereinbefore described to give a dinucleotideof formula VIII. The reaction between the nucleosides of formulae XIIand IX may be carried out in a solvent such as THF at ambienttemperature.

Nucleosides of formula XII can be prepared by treating nucleosides offormula II (salt forms of acids of formula XII) with acid usingconventional procedures.

Oligonucleotides having at least one internucleoside linkage of formulaI, for example an oligonucleotide having 2 to 200 nucleoside units, suchas an oligonucleotide analogue as hereinbefore described, may also beprepared by subjecting a nucleoside having a protected 5′ hydroxy groupand, at the 3′ position, a group of formula

where R¹ _(a) is as hereinbefore defined, R¹⁰ and R¹¹ are eachindependently an unsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀alkenyl, C₄ to C₁₀ cycloalkylalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkylgroup, or R¹⁰ is said group and R¹¹ is hydrogen, or R¹⁰ and R¹¹ togetherwith the nitrogen atom to which they are attached denote a five- tothirteen-membered heterocyclic ring, to a nucleoside coupling reactionwith a natural or synthetic nucleoside or oligonucleotide having a free5′ hydroxy group, to form an oligonucleotide precursor having aninternucleoside linkage of formula

where R¹ _(a) is as hereinbefore defined, and converting the precursorinto an oligonucleotide having an internucleoside linkage of formula

where R¹ _(a) is as hereinbefore defined and X is oxygen or sulphur byoxidising the precursor to give an oligonucleotide having aninternucleoside linkage of formula XV where X is oxygen or sulphurisingthe precursor to give an oligonucleotide having an internucleosidelinkage of formula XV where X is sulphur.

The reaction to form the precursor having a linkage of formula XIV maybe carried out in the presence of an amine-protonating coupling catalyst(activating agent) such as tetrazole or 5-(4-nitrophenyl)tetrazole. Thereaction may be carried out at −20 to 50° C., preferably at roomtemperature. The oxidation or sulphurisation of the resulting precursormay be effected by methods used for oxidation or sulphurisationrespectively of phosphite internucleoside linkages. Thus oxidation maybe effected by treatment with iodine and water, or with a hydroperoxidesuch as tert-butyl hydroperoxide, for example using conditions andprocedures known for oxidation of phosphite internucleoside linkages inoligonucleotide synthesis. Sulphurisation may be effected by treatmentwith sulphur in the presence of a tertiary amine in an organic solvent,usually carbon disulphide, by treatment with [3H]1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent) or by treatmentwith tetraethylthiuram, for example using procedures known forsulphurisation of phosphite internucleoside linkages.

Nucleosides having a protected 5′ hydroxy group and, at the 3′ position,a group of formula XIII, may be prepared by reacting a nucleoside havinga protected 5′ hydroxy group and, at the 3′ position, a group of formula

where R¹ _(a) is as hereinbefore defined and Z is halogen, with acompound of formula

where R¹⁰ and R¹¹ are as hereinbefore defined. The reaction may becarried out in an organic solvent, for example a halogenated hydrocarbonsuch as chloroform, in the presence of a tertiary nitrogen base such aspyridine, and at a temperature from −78° C. to 50° C., preferably from−30° C. to 25° C.

Nucleosides having a protected 5′ hydroxy group and, at the 3′ position,a group of formula XVI may be prepared by non-oxidative halogenation ofa nucleoside having a protected 5′ hydroxy group and, at the 3′position, a group of formula

The non-oxidative halogenation may be carried out by reaction with anon-oxidative halogenating agent, for example a halophosphorane such astriphenyldichlorophosphorane ordichlorotris(2,4,6-tribromophenoxy)phosphorane in the presence of abase, preferably a tertiary nitrogen base such as pyridine, in anorganic solvent, which may be pyridine but is preferably ahalohydrocarbon such as chloroform, at a temperature from −20° C. to 60°C., preferably from 0° C. to 50° C.

Nucleosides having a protected 5′ hydroxy group, and at the 3′ position,a group of formula XVIII may be prepared as described in WO 96/08503.

When the oligonucleotide having a linkage of formula XV is formed on asolid support, it may be treated to remove the 5′ protecting group andthe resulting 5′ hydroxy-terminated oligonucleotide subjected tosuccessive coupling cycles with a natural or synthetic nucleoside oroligonucleotide having a protected 5′ hydroxyl group and, at the 3′position, a group reactive with, or activatable to be reactive with, thefree 5′ hydroxy group on the deprotected oligonucleotide attached to thesolid support, until an oligonucleotide of the desired length isobtained. Thus the oligonucleotide having a linkage of formula XV may becoupled with a nucleoside or oligonucleotide having a 3′phosphoramidite, H-phosphonate, phosphodiester group or 3′ group offormula XIII and a protected 5′ hydroxyl group, to give a chain-extendedoligonucleotide which may in turn be further chain extended by furthersuch alternative reactions until an oligonucleotide of the desiredlength is obtained. Where a nucleoside or oligonucleotide having a 3′phosphoramidite, H-phosphonate or phosphodiester group is used, thecoupling reaction may be carried out using procedures known inoligonucleotide synthesis. Where a nucleoside having a 3′ group offormula XIII is used, the coupling reaction may be carried out ashereinbefore described. Thus, where a 3′ phosphoramidite or a 3′ groupof formula XIII is used, a coupling cycle involves an oxidation orsulphurisation while where a 3′H-phosphonate is used, oxidation orsulphurisation is effected after chain extension is complete, and wherea 3′ phosphodiester is used no oxidation is required.

The oligonucleotide analogues of the invention can be used intherapeutics, for example in the treatment of a human or other animalsuffering from a disease which is modulated by a protein, or in thetreatment of viruses such as influenza, herpes and HIV. Accordingly, thepresent invention also provides a pharmaceutical composition comprisingas active ingredient an oligonucleotide analogue of the invention.Optimum dosages and treatment schedules can readily be determined bythose skilled in the art. When administered to mammals of about 70 kgweight, the dose can be, for example, 0.01 to 1000 mg per day. It willgenerally be preferred to administer therapeutic agents in accordancewith the invention internally, for example orally, by inhalation,intravenously or intramuscularly. Other methods of administration, suchas transdermal, topical or inter-lesional methods, and by inclusion insuppositries, can also be useful. Use in conduction withpharmacologically acceptable carriers is preferred for some therapeutictreatments.

The oligonucleotide analogues according to the invention have asurprisingly high stability to degradation by nucleases. A very goodpairing with complementary nucleic acid strands, particularly of the RNAtype, is also observed. The oligonucleotide analogues according to theinvention are therefore particularly suitable for antisense technology,i.e. for inhibition of the expression of undesired protein products dueto the binding to suitable complementary nucleotide sequence in nucleicacids (see EP 0 266 099, WO 87/07300 and WO 89/08146). They can beemployed for the treatment of infections and diseases, for example byblocking the expression of bioactive proteins at the nucleic acid stage(for example oncogenes). The oligonucleotide analogues according to theinvention are also suitable as diagnostics and can be used as geneprobes for the detection of viral infections or of genetically relateddiseases by selective interaction at the single or double-strandednucleic acid stage. In particular—due to the increased stability tonucleases—diagnostic use is not only possible in vitro but also in vivo(for example tissue samples, blood plasma and blood serum). Usepossibilities of this type are described, for example, in WO 91/06556.

The novel dinucleotides of formula XI can be used as pharmaceuticals,for example as antiviral agents.

The pharmacologically active oligonucleotide analogues and dinucleotidesaccording to the invention can be used in the form of parenterallyadministrable preparations or of infusion solutions. Solutions of thistype are preferably isotonic aqueous solutions or suspensions, it beingpossible to prepare these before use, for example in the case oflyophilised preparations which contain the active substance on its ownor together with a carrier, for example mannitol. The pharmaceuticalpreparations can be sterilised and/or contain excipients, for examplepreservatives, stabilisers, wetting and/or emulsifying agents,solubilisers, salts for regulating the osmotic pressure and/or buffers.The pharmaceutical preparations, which if desired can contain furtherpharmacologically active substances such as, for example, antibiotics,are prepared in a manner known per se, for example by means ofconventional dissolving or lyophilising processes, and contain about0.1% to 90%, in particular from about 0.5% to about 30%, for example 1%to 5% of active substance(s).

This invention is illustrated by the following Examples.

Compounds used in the Examples, and precursors thereof, are prepared asfollows. All ³¹P data for these compounds and those of the Examples arefor ¹H decoupled.

To a solution of an aldehyde of formula XIII where R² is hydrogen, B¹ isI-thyminyl and R¹ is tert-butyl diphenylsilyl, prepared as described inWO 92/20823, (11.2 g 23 mmol) in anhydrous ethanol (120 ml) at roomtemperature is added NaBH₄ (865 mg, 23 mmol) portionwise over 5 minutes.After 1 hour, the reaction mixture is quenched with water, diluted withethylacetate (500 ml) and washed with water (2×50 ml). After backextraction of the aqueous phase, the combined organic phase is dried(MgSO₄) and concentrated to give Compound A as a white solid.

¹H nmr (CDCl₃, 400 MHz) δ9.10 (1H, s, NH) 7.65 (4H, d, Ar 4×CH ortho),7.40 (7H, m, Ar 4×CH meta, 2×CH para+H6) 6.13 (1H, t, H1′) 4.00 (1H, dd,H5′), 3.93 (1H, m, H4′) 3.82 (1H, dd, H5′), 3.62 (2H, m, CH ₂OH) 2.60(1H, m, H3′), 2.32 (1H, m, H2′), 2.12 (1H, m, H2′) 1.62 (3H, s, T-CH ₃)and 1.10 (9H, s, ^(t)Bu) ppm.

To a solution of Compound A (9 g, 18.1 mmol) in dry DMF (100 ml) at 0-5°C. is added 2,6-lutidine (4.25 ml, 36.5 mmol) followed bymethyltriphenoxyphosphonium iodide (9.45 g, 20.9 mmol). The resultingmixture is allowed to warm to room temperature. After 1 hour the mixtureis diluted (200 ml ethyl acetate) and washed with 0.1N NaS₂O₃ (2×20 ml),0.5N Hydrochloric acid (2×20 ml) and water (2×20 ml). Drying,concentration and purification by flash silica column chromatography(gradient elution chloroform:ethylacetate 20:1-7:1) gives Compound B asa white solid.

¹H nmr (CDCl₃, 400 MHz) δ10.2 (1H, s, NH) 7.66 (4H, d, 4×CH ortho), 7.40(7H, M, 4×CH meta, 2×CH para+H6) 6.19 (1H, t, H1′) 4.02 (1H, dd, H5′),3.82 (1H, m, H4′) 3.78 (1H, dd, HS′), 3.17 (1H, dd, CH ₂I) 3.10 (1H, ddCH ₂I), 2.68 (1H, m, H3′), 2.30 (1H, m, H2′), 2.23 (1H, m, H2′) 1.66(3H, s, CH₃-T), 1.10 (9H, s, tBu) ppm.

To a solution of ethyl (1,1-diethoxyethyl)phosphinate (5.51 g, 26.2mmol) in dry THF (170 ml), under argon, at −78° C. is added a solutionof potassium bis(trimethylsilyl)amide (34.6 ml, 0.75M solution intoluene) dropwise over 5 minutes. The resulting solution is stirred at−78° C. for 1 hour. A solution of Compound B (5.0 g, 8.25 mmol) in dryTHF (20 ml) is then added dropwise over 5 minutes. Stirring is continuedat −78° C. for 1 hour before warming to room temperature over 2 hours.Saturated aqueous ammonium chloride (50 ml) is then added and the wholemixture extracted with ethyl acetate (500 ml). The organic phase iswashed with saturated ammonium chloride (2×50 ml) and water (2×50 ml),dried over magnesium sulphate and concentrated. Purification by flashsilica column chromatography (eluant ethylacetate:ethanol 30:1) givesCompound C as a 1:1 mixture of diastereoisomers epimeric at phosphorous.

Trimethylsilylchloride (4.44 ml, 35 mmol) is added dropwise (2 minutes)at room temperature to a stirred solution of Compound C (2.4 g, 3.5mmol) in chloroform (25 ml) containing ethanol (1%) under argon. Afterstanding at −20° C. for 60 hours, a further portion oftrimethylsilylchloride (2.22 ml, 17.5 mmol) is added along with ethanol(200 μl) and the resulting solution stirred at room temperature for 7hours. Concentration and co-evaporation with chloroform (50 ml) gives awhite solid which is purified by flash silica column chromatography(eluant chloroform: ethanol 13:1) to give Compound D as a white solidisolated as a 1:1 mixture of diastereoisomers.

To a solution of Compound D (1.2 g, 2.1 mmol) in dry THF (30 ml)containing acetone (3.2 ml) is added in titanium (IV) isopropoxide (738μl, 2.48 mmol). After 15 minutes, concentration and passage through ashort column of silica (eluant ethyl acetate:ethanol 4:1) (500 ml) givesCompound E isolated as a mixture of 2 diastereoisomers.

³¹P nmr ¹H decoupled (CDCl₃, 162 MHz) δ55.0, 54.7 ppm.

Found: C, 57.7; H, 7.05; N, 4.05% C₃₂H₄₅N₂O₇PSi.2H₂O requires C, 57.8;H, 7.4; N, 4.2%

To a solution of Compound E (1.02 g, 1.62 mmol) and acetic acid (92 μl,16.1 mmol) in THF (10 ml) is added a solution of tetra-n-butyl ammoniumfluoride (1.63 ml, 1.0 Molar). After stirring at room temperature for 1hour, the mixture is concentrated and co-evaporated with chloroform (50ml). Purification by flash silica column chromatography (eluantchloroform:ethanol 9:1) gives Compound F isolated as a mixture of twodiastereoisomers.

Found: C, 45.55; H, 6.85; N, 6.4% C₁₆H₂₇N₂O₇P.1 2/3H₂O requires C, 45.7;H, 7.25; N, 6.6% ³¹P nmr ¹H decoupled (CDCl₃, 162 MHz) δ56.7, 56.5 ppm.

To a solution of Compound F (550 mg, 1.41 mmol) in pyridine (10 ml) isadded dimethoxytritychloride (958 mg, 2.83 mmol). After stirring at roomtemperature for 20 hours, concentration and purification by flash silicacolumn chromatography (eluant chloroform, methanol, triethylamine100:5:1) gives Compound G, isolated as a mixture of 2 diastereoisomers.

³¹P nmr ¹decoupled (CDCl₃, 162 MHz) δ54.9, 54.7 ppm.

To a solution of Compound G (0.85 g, 1.22 mmol) in anhydrous methanol(10 ml) is added sodium methoxide (1.5 ml 4.4N solution in methanol).After stirring for 16 hours at room temperature, concentration andpurification by flash silica column chromatography (gradientelution—chloroform, methanol, triethylamine 100:20:1-100:35:1), followedby further purification by passing a solution of the product in aqueous0.5% triethylamine through a Dowex 50W-X2 ion exchange column(triethylamine form) gives, after concentration, Compound H.

³¹P nmr ¹H decoupled (CD₃OD, 162 MHz) δ23.7 ppm.

Compound J is prepared as described in Example 98 of WO 96/08503.

In the formulae of Compounds K to M, T is 1-thyminyl, and DMTr isdimethoxytrityl.

To a solution of Compound H (500 mg, 0.71 mmol) anddicyclohexylcarbodiimide (189 mg, 0.92 mmol) in dry THF (5.4 ml) underargon at room temperature is added 3-hydroxy propionitrile (58 μl, 0.85mmol). The resulting solution is heated at 55° C. for 2 hours. Aftercooling, the mixture is filtered and diluted with ethyl acetate (20 ml)and washed with water and brine, dried over Na₂SO₄, filtered andconcentrated. The resulting product is taken up in dichloromethane (5ml) and filtered and concentrated, this process being repeated asrequired to remove dicyclohexyl urea, to give Compound K, isolated as amixture of diastereoisomers at phosphorus.

³¹P nmr (¹H decoupled) (CDCl₃, 162 MHz) δ37.4, 37.3 ppm.

To a solution of carefully dried Compound K (71 mg, 220 μmol) indeuterochloroform (0.5 ml) containing pyridine (80 μl, 1 mmol) is addeddichlorotriphenylphosphorane (113 mg, 350 μmol). The resulting mixtureis shaken to dissolve the phosphorane and then allowed to stand atambient temperature. The progress of the reaction is monitored by ³¹Pnmr. The product is Compound L. After 16 hours, additionaldichlorotriphenylphosphorane (28 mg, 87 μmol) is added. After anadditional 24 hours, ³¹P nmr shows the reaction to be 95% complete. Atotal 56 μl (0.67 mmol) of pyrrolidine is added in portions to the crudereaction mixture at −30° C. The resulting mixture is allowed to warm toroom temperature and then diluted with dichloromethane (20 ml), washedtwice with deionised water (2×10 ml), dried (Na₂SO₄) and concentrated.Purification by flash silica column chromatography gives Compound M.

EXAMPLES 1-13

In the following Examples, Compound H and Compound J are utilised asmonomers in the synthesis of oligonucleotide analogues. Unmodified5′-dimethoxytrityl substituted nucleoside H-phosphonates are alsoutilised as the commercially available triethylammonium salts andabbreviated as follows:

Tp=Thymidine H-phosphonate

Cp=N⁴-Benzoyl-deoxycytidine-H-phosphonate

Ap=N⁶-Benzoyl-deoxyadenosine-H-phosphonate

Gp=N²-Isobutyryl-deoxyguanosine-H-phosphonate.

The oligonucleotide syntheses are carried out manually in polypropylenesyringes with commercially available nucleoside (Tp, Cp, or Ap)derivatised long chain alkylamine controlled pore glass. The firstresidue shown at the 3′ end of the following Examples results from thiscommercial material. The syntheses are carried out on a 0.2 μmol scalein the 3′ to 5′ direction.

Example 1 Oligonucleotide Analogue 1

^(5′)TTT T*TC TCT CTC TCT^(3′) SEQ ID NO:3

where * is an internucleoside linkage of formula I, where X is an oxygenand R¹ is hydroxy, all other linkages being phosphodiester linkages.

The support (6.8 mg) is washed with dichloromethane and the5′-protecting group is removed by treatment with dichloroacetic acid andthe support is washed again in preparation for coupling. Where thecoupling is to be with an un-modified nucleoside H-phosphonate, thesupport is treated with a solution of the 5′-protected nucleosideH-phosphonate (30 mM for thymidine; 20 mM for cytidine) inpyridine-acetonitrile (1:1 v/v; 200 μl for thymidine, 300 μl forcytidine) in the presence of pivaloyl chloride (182 mM) also inpyridine-acetonitrile (1:1 v/v; 200 μl) for 1 minute.

To obtain the required oligonucleotide analogue normal H-phosphonate DNAsynthesis is followed except that monomer Compound H is substituted foran unmodified nucleoside H-phosphonate when the T* position is reachedin the synthesis and modified coupling and oxidation conditions are usedas described below.

When the monomer is a 3′-methylene phosphinate of formula II, then thesupport is treated twice with a solution of Compound H (60 mM) inpyridine-acetonitrile (1:1 v/v; 200 μl) in the presence of pivaloylchloride (121 mM) in pyridine-acetonitrile (1:1 v/v; 200 μl) for 30minutes per treatment, that is to say 2×30 minutes. Additional washsteps are carried out and the cycle is repeated with the removal of the5′-protecting group and the coupling of a fresh monomer, either CompoundH or a 3′-H-phosphonate, to the free 5′-hydroxyl group of the growingoligonucleotide chain.

These steps are repeated until the full length 15-mer precursor toOligonucleotide Analogue I has been prepared.

The steps of the synthetic cycle can be summarised as indicated below:

Step Reagent/Solvent Volume 1-wash dichloromethane 4 ml 2-deblock 2.5%(v/v) dichloroacetic acid in 4-5 ml dichloromethane 3-washdichloromethane 4 ml 4-wash pyridine-acetonitrile (1:1 v/v) 4 ml5-coupling i) Solution of Compound H 400 μl (60 mM) inpyridine-acetonitrile (1:1 v/v) (200 μl) treated with pivaloyl chloride(121 mM) in pyridine-acetonitrile (1:1 v/v; 200 μl) and allowed to reactfor 2 × 30 minutes. ii) monomers that are un-modified nucleoside 400 μl(T) H-phosphonates. The monomer solution 500 μl (C) (30 mM for Tp; 20 mMfor Cp) in pyridine- acetonitrile (1:1 v/v; 200 μl for Tp, 200 μl + 100μl pyridine for Cp) is treated with pivaloyl chloride (182 mM) inpyridine-acetonitrile (1:1 v/v; 200 μl) and allowed to react for 1minute. 6-wash pyridine-acetonitrile (1:1 v/v) 4 ml

7. Steps 1-6 are repeated with the appropriate monomers until theoligonucleotide precursor of Oligonucleotide Analogue I has beencompleted. After the final cycle of monomer addition has been carriedout, the support-mounted oligomer is treated with iodine (0.2M) inpyridine-water (8:2 v/v) during 1 hour at ambient temperature,optionally followed by further treatment with iodine (0.2M),pyridine-triethylamine-water (6:1:1 v/v) during 1 hour at ambienttemperature. This oxidises the internucleoside linkages. The support isthen washed with pyridine-acetonitrile (1:1 v/v), to remove traces ofiodine, followed by diethyl ether. The support is then air dried andtransferred to a plastic tube for treatment with aqueous ammoniasolution (30%) at 55° C. overnight. This removes the oligonucleotideanalogue from the support and cleaves the base protecting groups whilstleaving the 5′-0-dimethoxytrityl group intact. After this treatment theoligonucleotide analogue solution is subject to 5′-0-dimethoxytritylremoval on an oligonucleotide purification cartridge according tostandard procedures (Evaluating and Isolating SyntheticOligonucleotides, Applied Biosystems, 1992). The oligonucleotideanalogue is then purified by polyacylamide gel electrophoresis accordingto standard procedures.

MALDI-TOF mass spectroscopic analysis of Oligonucleotide Analogue I:

Calculated mass: 4424.3 Da

Found: 4423.8 Da

Example 2 Oligonucleotide Analogue 2

^(5′)TTT T*TC TCT CTC TCT^(3′) (SEQ ID NO:3)

where * is an internucleoside linkage of formula I where X is sulphurand R¹ is hydroxy. All other internucleoside linkages arephosphorothioate. The solid phase synthesis of the above oligonucleotideis carried out using the method of Example 1 above as far as the finalcycle of monomer addition. The treatment of the oligomer with iodineused in Example 1 is replaced by a sulphurisation reaction. Thus, afterthe final coupling step, the support mounted precursor toOligonucleotide Analogue 2 is treated with a solution of sulphur (5%w/v) in carbon disulphide-pyridine-triethylamine (10:10:1 v/v;2 ml) for1-18 hours at room temperature, following the procedure of A. Audrus andG. Zon, Nucleic Acids Research Symposium Series No. 20, 1988, 121. Atthe completion of the incubation period, the support is washed withpyridine-acetonitrile (1:1 v/v) and diethylether then air-dried. Theoligonucleotide analogue is removed from the support and protectinggroups removed as described in Example 1.

MALDI-TOF (negative mode) mass spectroscopic analysis of OligonucleotideAnalogue 2:

Calculated mass: 4649.3 Da

Found: 4642.8 Da

Example 3 Oligonucleotide Analogue 3

^(5′)TTT T*T^(Me)CTCT CTC TCT^(3′) SEQ ID NO:4

where * is an internucleoside linkage of formula I, where X is oxygenand R¹ is hydroxy. All other internucleoside linkages arephosphodiester. T^(me) is a thymidine residue having an α-methoxy grouppresent at the 2′-position rather than a hydrogen atom. Solid phasesynthesis of the above oligonucleotide is carried out, as described inExample 1, as far as nucleoside residue 11. Here, the support mountedprecursor to oligonucleotide analogue 3 is treated with a solution of5′-dimethoxytrityl-2′-O-methyl thymidine H-phosphonate (30 mM) inpyridine-acetonitrile (1:1 v/v; 200 μl) in the presence of pivaloylchloride (182 mM) also in pyridine-acetonitrile (1:1 v/v; 200 μl) for 1minute. The remainder of the synthesis is carried out as described inExample 1.

MALDI-TOF (negative mode) mass spectroscopic analysis of OligonucleotideAnalogue 3:

Calculated mass: 4454.4 Da

Found : 4448.6 Da

Example 4 Oligonucleotide Analogue 4

^(5′)TTT T*T^(Me)C TCT CTC TCT^(3′) (SEQ ID NO: 4)

where *is an internucleoside linkage of formula I, where X is sulphurand R¹ is hydroxy. All other internucleoside linkages arephosphorothioate. T^(Me) is a thymidine residue having an α-methoxygroup present at the 2′-position rather than a hydrogen atom. The solidphase synthesis is carried out as described in Example 3 and thesulphurisation and subsequent work-up and deblocking are carried out asdescribed in Example 2 above.

MALDI-TOF (negative mode) mass spectroscopic analysis of OligonucleotideAnalogue 4:

Calculated mass: 4679.3 Da

Found: 4664.1 Da

Example 5 Oligonucleotide Analogue 5

5′TTT* TT3′

where * is an internucleoside linkage of formula I where X is oxygen andR¹ is hydroxy. All other internucleoside linkages are phosphodiesterlinkages.

This is prepared using the procedure of Example 1, except that CompoundH and Compound Tp are the only nucleoside monomers used.

MALDI-TOF Mass Spectroscopic Analysis:

Calculated Mass: 1457.1 Da

Found: 1457.7 Da

Example 6 Oligonucleotide Analogue 6

5′TTT TT*C TCT CTC TCT3′ SEQ ID NO:5

where * is an internucleoside linkage of formula I where X is oxygen andR¹ is hydroxy. All other internucleoside linkages are phosphodiesterlinkages.

This is prepared using the general procedure of Example 1.

Example 7 Oligonucleotide Analogue 7

5′T*T*T* T*T*C TCT CTC TCT3′ SEQ ID NO:6

Where * is an internucleoside linkage of formula I where X is oxygen andR¹ is hydroxy. All other linkages are phosphodiester linkages.

This is prepared using the general procedure of Example 1, butintroducing five nucleoside residues from Compound H.

Example 8 Oligonucleotide Analogue 8

5′CGA CTA TGC AT*T T*TC3′ SEQ ID NO:7

Where * is an internucleoside linkage of formula I where X is oxygen andR¹ is hydroxy. All other linkages are phosphodiester linkages.

This is prepared using the general procedure of Example 1, butintroducing two nucleoside residues from Compound H. When the sequencerequires the use of an A or G nucleoside then the coupling step of themethod of Example 1 is modified as follows:

i) 30 mM Ap or 30 mM Gp in pyridine-acetonitrile (1:1 v/v; 200 μl of Apor Gp) is treated with pivaloyl chloride (182 mM) inpyridine-acetonitrile (1:1 v/v; 200 μl) and allowed to react for 1minute.

MALDI-TOF mass spectroscopic analysis of Oligonucleotide Analogue 8:

Calculated mass: 4514.1 Da

Found: 4517.3 Da

Example 9 Oligonucleotide Analogue 9

5′GCG T*T*T* T*T*T* T*T*T* T*GC G3′ SEQ ID NO:8

Where * is an internucleoside linkage of formula I where X is oxygen andR¹ is hydroxy. All other linkages are phosphodiester linkages.

This is prepared using the general procedures of Examples 1 and 8, butintroducing ten nucleoside residues from Compound H.

Example 10 Oligonucleotide Analogue 10

5′TTT TT*C* TCT CTC TCT3′ SEQ ID NO:9

Where * is an internucleoside linkage of formula I where X is oxygen andR¹ is hydroxy. All other linkages are phosphodiester linkages.

This is prepared using the general procedure of Example 1, except thatwhen the C* residue is to be introduced monomer Compound J is used as adirect replacement for Compound H.

Example 11 Oligonucleotide Analogue 11

5′TTT TTT TTT TTT TTT T*T*T* T3′ SEQ ID NO:10

Where * is an internucleoside linkage of formula I where X is oxygen andR¹ is hydroxy. All other linkages are phosphodiester linkages.

This is prepared using the general procedure of Example 1, butintroducing three nucleoside residues from Compound H.

Example 12 Oligonucleotide Analogue 12

5′T*T*C* T*C*G CCC GCT CC*T* C*C*T* C*C3′ SEQ ID NO :11

Where is an internucleoside linkage of formula I where X is sulphur andR¹ is hydroxy. All other linkages are phosphorothioate.

This is prepared using the general procedure of Example 2. G residuesare introduced as described in Example 8, and C* residues are introducedas described in Example 10.

Example 13 Oligonucleotide Analogue 13

5′T*T*C* T*C*G CTG GTG AGT* T*T*C* A3′ SEQ ID NO:12

Where * is an internucleoside linkage of formula I where X is sulphurand R¹ is hydroxy. All other linkages are phosphorothioate.

This is prepared using the general procedure of Example 12. A residuesare introduced as described in Example 8.

Example 14

Oligonucleotide Analogue 1 having the sequence 5′TTT tTC TCT CTC TCT3′(SEQ ID NO:13) where t represents a nucleoside unit derived fromCompound M, is prepared by solid phase phosphoramidite oligonucleotidesynthesis as described in ‘Oligonucleotides and Analogues A practicalApproach’ ed F. Eckstein, IRL press 1991, except that Compound M is usedinstead of the usual 3 ′-phosphoramidite-substituted nucleoside at theappropriate point in the synthesis, so that an oligonucleotide precursorhaving an internucleoside linkage of formula XIV where R¹ _(a) is—OCH₂CH₂CN is formed and then oxidised to an oligonucleotide having aninternucleoside linkage of formula XV where R¹ _(a) is —OCH₂CH₂CN and Xis oxygen in the standard oxidation step, this oligonucleotide beingcoupled further to give Oligonucleotide Analogue 1.

13 1 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic Oligonucleotide 1 tcccgcctgt gacatgcatt 20 2 20 DNA ArtificialSequence Description of Artificial Sequence Synthetic Oligonucleotide 2gttctcgctg gtgagtttca 20 3 15 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Oligonucleotide 3 tttttctctc tctct 15 4 15DNA Artificial Sequence Description of Artificial Sequence SyntheticOligonucleotide 4 tttttctctc tctct 15 5 15 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Oligonucleotide 5tttttctctc tctct 15 6 15 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Oligonucleotide 6 tttttctctc tctct 15 7 15DNA Artificial Sequence Description of Artificial Sequence SyntheticOligonucleotide 7 cgactatgca ttttc 15 8 16 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Oligonucleotide 8gcgttttttt tttgcg 16 9 15 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Oligonucleotide 9 tttttctctc tctct 15 1019 DNA Artificial Sequence Description of Artificial Sequence SyntheticOligonucleotide 10 tttttttttt ttttttttt 19 11 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Oligonucleotide 11ttctcgcccg ctcctcctcc 20 12 19 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Oligonucleotide 12 ttctcgctgg tgagtttca 1913 15 DNA Artificial Sequence Description of Artificial SequenceSynthetic Oligonucleotide 13 tttttctctc tctct 15

What is claimed is:
 1. An oligonucleotide analogue having 10 to 200natural and/or synthetic nucleoside units linked by internucleosidelinkages, and a 3′-terminal hydroxyl group, wherein at least one of theinternucleoside linkages is of formula

where the indicated methylene group is attached to a 3′ carbon atom of anucleoside, the indicated oxygen atom is attached to a 5′-carbon atom ofan adjacent nucleoside, R¹ is hydrogen, a group of formula R¹ _(a), or—NR¹ _(b)R¹ _(c) wherein R¹ _(a) is a C₁ to C₁₀ alkyl, C₂ to C₁₀alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl groupor a C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀aryl or C₇ to C₁₃ aralkyl group bearing one or more terminalsubstituents; and R¹ _(b) and R¹ _(c) are each independently anunsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ toC₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group; and x isoxygen or sulfur.
 2. An oligonucleotide analogue according to claim 1,which has 15 to 40 nucleoside units.
 3. An oligonucleotide analogueaccording to claim 1, which has 15 to 25 nucleoside units.
 4. Anoligonucleotide analogue according to claim 1 in which all theinternucleoside linkages are of formula I.
 5. An oligonucleotideanalogue according to claim 1 in which up to 75% of the internucleosidelinkages are of formula I.
 6. An oligonucleotide analogue according toclaim 5, in which up to 50% of the internucleoside linkages are offormula I.
 7. An oligonucleotide analogue according to claim 6, in whichup to 25% of the internucleoside linkages are of formula I.
 8. Anoligonucleotide analogue of claim 1, wherein at least two consecutiveinternucleoside linkages are of formula I.
 9. An oligonucleotideanalogue of claim 1, in which internucleoside linkages of formula Ialternate with other internucleoside linkages.
 10. An oligonucleotideanalogue of claim 1, wherein the remaining internucleoside linkages arephosphodiester, phosphorothioate or phosphorodithioate linkages or amixture of two or more thereof.
 11. An oligonucleotide analogueaccording to claim 10, in which the remaining internucleoside linkagesare phosphorothioate linkages.
 12. An oligonucleotide analogue accordingto claim 10, further comprising a region having phosphodiester and/orphosphorothioate and/or phosphorodithioate internucleoside linkagesbetween two regions having internucleoside linkages of formula I or amixture thereof with phosphorothioate or phosphodiester linkages.
 13. Anoligonucleotide analogue according to claim 1, in which at least onenucleoside is modified at the 2′ position thereof.
 14. Anoligonucleotide analogue according to claim 13, in which at least onenucleoside has a halogen atom or a group of formula —OR² at the 2′position, where R² is a C₁ to C₁₀ aliphatic group.
 15. Anoligonucleotide analogue according to claim 14, in which at least onenucleoside has a group —OR² at the 2′ position, where R² is anunsubstituted or substituted C₁ to C₁₀ alkyl group.
 16. Anoligonucleotide analogue according to claim 15, in which R² is C₁ to C₄alkyl, C₁ to C₄ alkoxy-substituted C₁ to C₄ alkyl or a group of formula—(CH₂CH₂O)—_(n)R³ where R³ is methyl or ethyl and n is 2 to
 4. 17. Anoligonucleotide analogue according to claim 16, in which R² is methyl,ethyl, methoxyethyl, ethoxyethyl or a group of formula —(CH₂CH₂O)—₃CH₃.18. An oligonucleotide analogue according to claim 13, in which at leasttwo consecutive nucleosides are modified at the 2′ position and arelinked by phosphodiester internucleoside linkages and/or there is alinkage of formula I between a nucleoside unmodified at the 2′ positionand a 5′ carbon atom of a nucleoside modified at the 2′ position.
 19. Anoligonucleotide according to claim 1, in which R¹ _(a), R¹ _(b) or R¹_(c) as alkyl, alkenyl, cycloalkyl, aryl, or aralkyl are unsubstitutedor substituted by hydroxy, C₁ to C₄ alkoxy, halogen, cyano, tri(C₁-C₁₅hydrocarbyl)silyl or primary, secondary or tertiary amino.
 20. Anoligonucleotide analogue according to claim 1 in which R¹ is hydrogen,hydroxy, O⁻, SH, S⁻, an unsubstituted or substituted C₁ to C₄ alkyl orphenyl group, a group of formula —OR¹ _(a), where R¹ _(a) is anunsubstituted or substituted C₁ to C₄ alkyl, C₃ to C₅ alkenyl, C₅ to C₈cycloalkyl or C₇ to C₉ aralkyl group, or a group of formula —SR¹ _(a)where R¹ _(a) is an unsubstituted or substituted C₁ to C₄ alkyl orphenyl group.
 21. An oligonucleotide analogue according to claim 20, inwhich R¹ is hydrogen, hydroxy, O⁻, SH, S⁻, methoxy, ethoxy or2-cyanoethoxy.
 22. An oligonucleotide analogue according to claim 1which is complementary to a region of mRNA for human c-raf kinase. 23.An oligonucleotide analogue according to claim 22 in which thenucleoside sequence is 5′-TCC CGC CTG TGA CAT GCA TT-3′ (SEQ ID NO:1).24. An oligonucleotide analogue according to claim 1, which iscomplementary to a region of mRNA for human PKC-α.
 25. Anoligonucleotide analogue according to claim 24, in which the nucleosidesequence is 5′-GTT CTC GCT GGT GAG TTT CA-3′ (SEQ ID NO:2).
 26. A methodof preparing an oligonucleotide analogue having at least oneinternucleoside linkage of formula I

where R¹ and X are as defined in claim 1, which comprises (i) carryingout a coupling reaction or successive coupling reactions between (A) anatural or synthetic nucleoside or oligonucleotide having a 5′-hydroxylgroup and (B) a natural or synthetic nucleoside or dinucleotide havingat the 3′-position thereof a group reactive with said 5′-hydroxyl groupuntil an oligonucleotide having the desired number of nucleosides isobtained, in at least one of said coupling reactions (B) being anucleoside of formula

where B¹ is a nucleoside base radical, R⁴ is a hydroxy-protecting group,R⁵ is hydrogen, hydroxy or a 2′ modifying atom or group, M⁺ is a metalor unsubstituted or substituted ammonium ion or a cation of aheterocyclic base, and X is oxygen or sulphur, and being reacted with(A) in the presence of a sterically hindered organic acid halide oranhydride to form an oligonucleotide analogue having a phosphinateinternucleoside linkage of formula

where X is oxygen or sulphur, and (ii)(a) oxidising the phosphinatelinkage or (b) sulphurising the phosphinate linkage, or (c) reacting thephosphinate linkage with a compound of formula R¹ _(a)Y where R¹ _(a) isas defined in claim 1 and Y is a leaving atom or group or (d) oxidisingand reacting the phosphinate linkage with an alcohol of formula R¹_(a)OH or an amine of formula R¹ _(b)NH₂ or R¹ _(b)R¹ _(c) NH where R¹_(a), R¹ _(b) and R¹ _(c) are as defined in claim 1, or (e) silylatingthe phosphinate linkage and reacting the silylated linkage with athioalkylating or thioarylating agent to give a phosphinate linkage offormula I where R¹ is —SR¹ _(a) where R¹ _(a) is as defined in claim 1.27. A method according to claim 26, in which the oligonucleotideanalogue is further reacted to replace the protecting group R⁴ byhydrogen or, where R⁴ is on a terminal nucleoside in the oligonucleotideanalogue, by a 5′ modifying group.
 28. A method according to claim 26which comprises the step of: carrying out the successive couplingreactions (i) and step (ii) of claim 26 with the nucleoside oroligonucleotide (A) attached to the solid support; (iii) detaching theoligonucleotide from the solid support and removing protecting groups togive an oligonucleotide having a terminal 5′ free hydroxyl group; and(iv) optionally reacting the 5′ free hydroxyl group to introduce amodifying group at the terminal 5′ position.
 29. A method according toclaim 26 wherein, in formula II, B¹ is a pyrimidine base, R⁴ is amethoxytrityl, dimethoxytrityl or tris tert-butyltrityl group, R⁵ ishydrogen and M⁺ is an unsubstituted ammonium, mono-, di- or tri-C₁-C₁₀alkyl- or hydroxyalkyl-ammonium ion or a cation of a heterocyclic base.30. A method according to claim 26 wherein the nucleoside of formula IIis a stereoisomer having the formula:


31. A method according to claim 26 wherein the coupling reaction of thenucleoside of formula II with (A) in the presence of a stericallyhindered organic acid halide is carried out in the presence of aheterocyclic base having a tertiary nitrogen atom in the ring or anoxide thereof.
 32. A method according to claim 26 wherein oxidation(ii)(a) is effected by treatment with iodine and water, or withtert-butyl hydroperoxide.
 33. A method according to claim 26 whereinsulphurisation (ii)(b) is effected by treatment with sulphur in thepresence of a tertiary amine in an organic solvent.
 34. A methodaccording to claim 26 wherein the reaction (ii)(c) of the phosphinateinternucleoside linkage of formula 1A with a compound of formula R¹_(a)Y where R¹ _(a) is alkyl, cycloalkyl or aralkyl as defined in claim1 and Y is halogen is carried out in the presence of a strong base. 35.A method according to claim 26 wherein the reaction (ii)(c) of thelinkage of formula IA with a compound of formula R¹ _(a)Y, where R¹_(a)Y an alkenyl or aryl halide or triflate, is carried out in thepresence of a palladium catalyst.
 36. A method according to claim 26wherein the oxidative reaction (ii)(d) of the phosphinateinternucleoside linkage of formula IA with an alcohol of formula R¹_(a)OH is carried out by reaction with an oxidant in the presence of thealcohol R¹ _(a)OH and a base.
 37. A method according to claim 36, inwhich the oxidant is iodine, carbon tetrachloride orbromotrichloromethane, and the base is pyridine.
 38. A method accordingto claim 26 wherein the oxidative reaction (ii)(d) of the phosphinateinternucleoside linkage of formula IA is effected with an amine offormula R¹ _(b)NH₂ or R¹ _(b)R¹ _(c)NH where R¹ _(b) and R¹ _(c) are asdefined in claim 1 and carbon tetrachloride or bromotrichloromethane oriodine to give an oligonucleotide analogue of the invention in which R¹is —NHR¹ _(b) or —NR¹ _(b)R¹ _(c) respectively.
 39. A method accordingto claim 26 wherein the reaction (ii)(e) of the phosphinate linkage offormula IA is carried out by silylating the linkage using atrialkylsilyl halide and a base, and reacting the silylated linkage witha thioalkylating or thioarylating agent.
 40. A method according to claim39, in which the thioalkylating or thioarylating agent is athiosulphonate of formula ArSO₂SR¹ _(a) where R¹ _(a) is as defined inclaim 1 and Ar is an aromatic group.
 41. A method according to claim 26wherein when an oligonucleotide analogue having the desired number ofnucleosides has been synthesised on a solid support, it is detached fromthe solid support, by treatment with concentrated aqueous ammonia,before or after treatment to remove hydroxy-protecting groups.
 42. Amethod according to claim 26 wherein hydroxy-protecting groups areremoved by treatment with an aqueous organic acid.
 43. A methodaccording to claim 26 wherein the nucleoside of formula II is replacedby a dinucleotide having the formula:

where B¹ is a nucleoside base radical, R¹ is hydrogen, hydroxy, O⁻,thiol, S⁻, —NH₂ or a group of formula R¹ _(a), —OR¹ _(a), —SR¹ _(a),—NHR¹ _(b) or —NR¹ _(b)R¹ _(c) where R¹ _(a) is an unsubstituted orsubstituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆to C₁₀ aryl or C₇ to C₁₃ aralkyl group, R⁴ is a hydroxy-protecting groupand R⁵ is hydrogen, hydroxy or a 2′ modifying atom or group, B² is anucleoside base radical, R⁶ is hydrogen, hydroxy or a 2′ modifying atomor group and R⁷ is a group reactive with, or activatable to be reactivewith, a 5′ hydroxyl group in a nucleoside.
 44. A method according toclaim 43, in which R⁷O in formula VIII is a H-phosphonate group, aphosphoramidite group or a phosphodiester group.
 45. A dinucleotide offormula

where B¹ is a nucleoside base radical, B² is a nucleoside base radical,R¹ is hydrogen, hydroxy, O⁻, thiol, S⁻, —NH₂ or a group of formula R¹_(a), —OR¹ _(a), —SR¹ _(a), —NHR¹ _(b) or —NR¹ _(b)R¹ _(c) where R¹ _(a)is an unsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl,C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group, R⁴ is ahydroxy-protecting group, R⁵ is hydrogen, hydroxy or a 2′ modifying atomor group, and R⁶ is hydrogen, hydroxy or a 2′ modifying atom or groupexcept that where R¹ is other than hydrogen at least one of R⁵ and R⁶ isa 2′ modifying atom or group and R⁹ is a group reactive with, oractivatable to be reactive with, a 5′ hydroxyl group in a nucleoside ora hydroxy-protecting group.